Leptomeningeal anastomoses play a critical role in regulating reperfusion following cerebrovascular obstruction; however, methods to evaluate their temporospatial remodeling remains under investigation.

We combined arteriole-specific vessel painting with histological evaluation to assess the density and diameter of inter-collateral vessels between the middle cerebral artery and anterior cerebral artery (MCA-ACA) or posterior cerebral artery (MCA-PCA) in a murine model of permanent middle cerebral artery occlusion (pMCAO).

While the overall density was not influenced by pMCAO, the size of MCA-ACA and MCA-PCA vessels had significantly increased 2 days post-pMCAO and peaked by 4 days compared to the un-injured hemisphere. Using a combination of vessel painting and immunofluorescence, we uniquely observed an induction of cellular division and a remodeling of the smooth muscle cells within the collateral niche following post-pMCAO on whole mount tissue sections. Vessel painting was also applied to pMCAO-injured Cx3cr1^GFP mice, in order to identify the spatial relationship between Cx3cr1-positive peripheral-derived monocyte/macrophages and the vessel painted collaterals. Our histological findings were supplemented with analysis of cerebral blood flow using laser Doppler imaging and behavioral changes following pMCAO.

Compared to polyurethane and latex methods for collateral labeling, this new method provides detailed cell-type specific analysis within the collateral niche at the microscopic level, which has previously been unavailable.

This simple and reproducible combination of techniques is the first to dissect the temporospatial remodeling of pial collateral arterioles. The method will advance investigations into the underlying mechanisms governing the intricate processes of arteriogenesis.